Study design – We retrospectively reviewed the positive fungal cultures of clinically significant specimens recovered in the Clinical Microbiology Laboratory at Northwestern Memorial Hospital, a 900-teaching hospital in Chicago, between January 1, 2013, and December 31, 2017. Specimen types include abscess, body fluids, bronchoalveolar lavage, endotracheal aspirate, sputum, skin, tissues, and wound. Information collected includes the fungal smear result, culture starting date, growth detection date, and species identification. Time to culture positivity was counted in days from when the culture was started to the time when fungal growth was first detected.
Fungal stain and culture – Microscopic analysis of the fungal smear was performed on all specimens with a wet mount in 20% KOH and with Calcofluor-white staining. The smear of sterile body fluids was prepared with cytospin centrifugation. Clinical specimens were cultured on inhibitory mold agar plates and brain and heart infusion agar plates. Plates were incubated at 30 °C in ambient air for 4 weeks. All plates were examined 3 times a week during the 2 weeks of incubation and then once a week thereafter. The time to detection of fungal growth was counted in days from when the culture was started to the time when fungal growth was first detected.
ITS PCR – During the study period, infectious disease physicians had access to broad-range ITS PCR for culture-negative patients with high clinical suspicion of fungal infections. The test was offered as an add-on test performed using the residual sample after the specimen was cultured. Only specimens from sources free from colonization by fungal organisms were tested. When ITS PCR became positive, identification was determined with sequencing.
DNA extraction for ITS PCR was performed in the clinical microbiology laboratory at Northwestern Memorial Hospital using established protocols. Briefly, 200 μL of fluid or 0.03 grams of solid were used for nucleic acid extraction. DNA was extracted with the QIAamp DNA Mini Kit (Qiagen Sciences, LLC, Louisville, KY). PCR amplification of the ITS region was performed with primers ITS1 5’- TCCGTA GGTGAACCTGCG G- 3’ and ITS4 5’- TCCTCCGCTTATGATATG C - 3’ as described preciously (15). Each PCR reaction (50 µL) consisted of 10x Buffer, Taq polymerase (Invitrogen Platinum taq DNA polymerase high fidelity), 50mM MgSO¬4, dNTP, DNase/RNase-free H20, and 4 µL DNA template. To ensure the quality of the nucleic acid extraction and PCR process, 3 sets of controls were included with each PCR run. An internal extraction/inhibition control using primers to the Beta globin gene was included to account for false-negative results. Sterile water and Candida albicans ATCC® 10231 were used as negative and positive DNA controls, respectively. Patient samples and controls were run on a C1000 Touch Thermal Cycler (Bio-Rad Laboratories, Hercules, CA) with the following conditions: denaturation 95 °C for 5 min, 34 cycles of denaturation for 30 s, annealing at 56 °C for 30 s, and extension at 68 °C for 1 min. Following amplification, gel electrophoresis was performed to determine whether fungal nucleic acid was present in the specimen and whether the controls performed as expected. The PCR product was purified using the Qiagen QIAquick PCR Purification Kit (Qiagen, Valencia, CA) prior to sequencing. Sequencing was performed on the ABI 3500 Sequencer (Thermal Fisher Scientific, Waltham, MA) on the eluted DNA. Identification was obtained by BALST search against the National Center for Biotechnology Information’s GenBank (https://blast.ncbi.nlm.nih.gov/Blast.cgi). An identification score of >99% was used to determine the identification. To be consistent with the report of culture results, species identification was reported for yeast isolates and dimorphic fungi; genus identification was reported for other fungal species unless species identification was requested by clinicians. The limit of detection of the ITS PCR assay at NMH is approximately 1000 CFU/mL.
Medical record review – Electronic medical records were reviewed to determine the clinical significance of the organisms recovered by culture after 14 days of incubation and the organisms detected by ITS PCR. A result was determined to be clinically significant when the treating physician’s diagnosis based on the test result was recorded in the patient chart and the patient was treated for the identified organism.